万寿菊番茄红素β-环化酶基因及其启动子克隆和功能分析

Cloning and Functional Analysis of Lycopene β-cyclase Promoter of Marigold(Tagetes erecta)

【目的】克隆万寿菊番茄红素β-环化酶基因和其启动子,进行生物学信息分析,并预测启动子功能,为万寿菊类胡萝卜素的代谢机制和叶黄素含量的调控提供参考依据。【方法】通过RT-PCR技术从万寿菊总RNA中克隆得到TeLCYb的cDNA序列;应用生物学方法分析其DNA序列及其编码蛋白质序列特征,使用DNAMAN和在线软件Box Shade对其氨基酸序列同源性比对分析,并利用MEGA6.0构建进化树,分析其亲缘关系;根据其cDNA序列利用FPNI-PCR法克隆其启动子序列,利用Plant Care在线数据库分析万寿菊TeLCYb启动子调控元件,构建启动子缺失表达载体pTeLCYb(-1969)∷GUS和pTeLCYb(-1140)∷GUS,利用农杆菌介导法侵染烟草,以GUS为报告基因研究不同调控元件的活性。【结果】从万寿菊中成功克隆TeLCYb,生物信息学分析发现,其全长共1 865 bp,开放阅读框为1 527 bp,编码508个氨基酸,氨基酸序列同源比对结果表明其与西洋蒲公英和菊花的同源性最高,且具有LCYb蛋白质特有的保守功能位点和特征多肽序列,在进化上与菊科单独聚为一枝。在已知基因序列的基础上获得长度为1 806 bp的TeLCYb启动子序列,生物学信息分析表明:该启动子除包含核心启动子元件TATA-box、CAAT-box外,还含有多种光应答元件和激素响应元件,其中光响应元件有13个,激素响应元件有5个,此外还具有MYBHv1结合位点元件、耐热响应元件、生理调控作用元件等顺式调控元件。GUS化学组织染色结果显示不同长度启动子均能够驱动GUS在茎、叶、花药及柱头中表达,但不同组织GUS染色蓝色斑点深度不同,其中花器官中花药和柱头中GUS酶活性最强;相对于启动子pTeLCYb(-1969),pTeLCYb(-1140)启动子还能够驱动GUS在根、叶和花萼中特异性表达,且各组织中GUS酶活性明显强于启动子pTeLCYb(-1969)的GUS化学组织染色结果。【结论】不同长度TeLCYb启动子均能驱动下游GUS的表达,但启动子的作用部位和作用强度存在差异,推测在ATG上游1 140 bp和164 bp区间的光响应元件可能具有增强子的功能,而全长启动子特有的激素响应元件和热响应元件可能具有抑制或降低启动子功能的作用。

【Objective】The objective of this study is to clone Lycopene β-cyclase enzyme（LCYB） gene and its promoter from marigold（Tagetes erecta）, to analyze their bioinformatics characters and predict the promoter function. This study provides a reference for the regulation of the metabolic mechanism of the carotenoids and the control of the lutein concentration in marigold.【Method】The cDNA sequence of TeLCYb gene was cloned using RT-PCR method. Bioinformatics tools were applied to analyze both the TeLCYb gene sequence and the characteristics of its encoded protein sequence. DNAMAN and online software Box Shade were used to make multiple sequence alignments between the TeLCYb amino acid sequence and their homologous sequences, and MEGA6.0 was used to constructed phylogenetic tree of homologous species. The promoter sequence was cloned according to its cDNA sequence using FPNI-PCR. Thereafter, two different 5′ UTR deletion mutants of the TeLCYb gene promoter were amplified by PCR, and then were inserted into the vector V152 to construct promoter-GUS fusion genes expression vectors, named pTeLCYb（-1969）：：GUS and pTeLCYb（-1140）：：GUS, respectively, and then were transformed into tobacco by agrobacterium tumefaciens-mediated transformation of tobacco, respectively. And the promoter activities were quantitatively estimated using GUS report gene.【Result】The TeLCYb was successfully cloned from marigold（Tagetes erecta）. The bioinformatics analysis showed that its full length was 1 865 bp with an open reading frame（ORF） of 1 527 bp length, encoding 508 amino acids. Homology analysis showed that the deduced TeLCYb protein was highly homologous to other LCYb proteins from Chrysanthemum morifolium, Dendranthema lavandulifoliu and TeLCYb protein has typical protein special elements of LCYb protein. Phylogenetic analysis also indicated that TeLCYb was clustered into the branch with asteracea. A 1 806 bp promoter sequence was obtained via FPNI-PCR. Bioinformatics analysis of promoter sequence revealed that this fragment contained some promoter core elements, such as TATA-box and CAAT-box, and 13 light responsive elements and 5 hormones responses elements. Meanwhile, MYB transcription factor binding elements and heat-resistant response elements, physiological control elements and cis-acting regulatory elements were found in this sequence. Functional characterization of promoters by constructing pTeLCYb（-1969）：：GUS and pTeLCYb（-1140）：：GUS plant expression vectors and transforming into tobacco respectively showed that both promoters were active in stems, petals, anthers and capitals. But there were significant differences of GUS enzyme activity among plant tissues, and blue spots found in anthers and capital were significantly stronger than that of petals. Compared with the expression level of pTeLCYb（-1969）：：GUS, the shorter sequence of pTeLCYb（-1140） led to a significant increase in the activity of GUS in the transgenic leaves and root and sepals were also found GUS enzyme activity. 【Conclusion】 Different length of promoters can drive the expression of GUS gene, but there are differences in the sites and intensity where promoters functions, we hypothesize that a positive regulatory factor of light response elements maybe exist between 1 140 bp and 164 bp area of 5′ upstream to ATG, while hormone response elements and the heat stress response element may suppress or reduce the promoter function.